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Accepted Manuscript
Norbornene probes for the study of cysteine oxidation
Lisa J. Alcock, Kyle D. Farrell, Mawey T. Akol, Gregory H.
Jones, Matthew M.Tierney, Holger B. Kramer, Tara L. Pukala, Gonçalo
J.L. Bernardes, Michael V.Perkins, Justin M. Chalker
PII: S0040-4020(17)31147-X
DOI: 10.1016/j.tet.2017.11.011
Reference: TET 29091
To appear in: Tetrahedron
Received Date: 3 September 2017
Revised Date: 2 November 2017
Accepted Date: 3 November 2017
Please cite this article as: Alcock LJ, Farrell KD, Akol MT,
Jones GH, Tierney MM, Kramer HB, PukalaTL, Bernardes GonçJL,
Perkins MV, Chalker JM, Norbornene probes for the study of cysteine
oxidation,Tetrahedron (2017), doi: 10.1016/j.tet.2017.11.011.
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https://doi.org/10.1016/j.tet.2017.11.011
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Norbornene probes for the study of cysteine oxidation Lisa J.
Alcock, Kyle D. Farrell, Mawey T. Akol, Gregory H. Jones, Matthew
M. Tierney, Holger B. Kramer, Tara L. Pukala, Gonçalo J. L.
Bernardes, Michael V. Perkins, Justin M. Chalker*
Leave this area blank for abstract info.
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Graphical Abstract To create your abstract, type over the
instructions in the template box below. Fonts or abstract
dimensions should not be changed or altered.
Norbornene probes for the study of cysteine oxidation Lisa J.
Alcock, Kyle D. Farrell, Mawey T. Akol, Gregory H. Jones, Matthew
M. Tierney, Holger B. Kramer, Tara L. Pukala, Gonçalo J. L.
Bernardes, Michael V. Perkins, Justin M. Chalker*
Leave this area blank for abstract info.
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Tetrahedron
journal homepage: www.e lsevier .com
Norbornene probes for the study of cysteine oxidation
Lisa J. Alcock,a Kyle D. Farrell,a Mawey T. Akol,a Gregory H.
Jones,b Matthew M. Tierney,c Holger B. Kramer,d Tara L. Pukala,e
Gonçalo J. L. Bernardes,f,g Michael V. Perkins a and Justin M.
Chalkera,∗ aFlinders University, College of Science and
Engineering, Sturt Road, Bedford Park, South Australia 5042,
Australia bCalifornia Institute of Technology, Division of
Chemistry and Chemical Engineering, 1200 East California Boulevard,
Pasadena, CA 91125, USA c The University of North Carolina at
Chapel Hill, Department of Chemistry, Chapel Hill, NC 27599, USA d
Imperial College London, MRC London Institute of Medical Sciences,
Hammersmith Hospital Campus, Du Cane Road, London, W12 0NN, UK e
The University of Adelaide, School of Physical Sciences, Adelaide,
South Australia 5005, Australia f University of Cambridge,
Department of Chemistry, Lensfield Road, Cambridge CB2 1EW, UK g
Instituto de Medicina Molecular, Faculdade de Medicina,
Universidade de Lisboa, Avenida Professor Egas Moniz, 1649-028,
Lisboa, Portugal
Introduction
Figure 1: Cysteine reacts with hydrogen peroxide to form a
sulfenic acid.
Cysteine oxidation is a critical aspect of redox homeostasis,
protein folding, and intracellular signaling.1-3 This oxidation can
occur by reaction of the thiolate side chain of cysteine with
hydrogen peroxide and other reactive oxygen or reactive nitrogen
species generated in cells by the mitochondria and various oxidase
enzymes.4-6 The immediate product of the reaction of cysteine with
hydrogen peroxide is cysteine sulfenic acid (1, Fig. 1). Cysteine
sulfenic acid may be the first product formed during the scavenging
of reactive species during oxidative stress, but it is also a
critical determinant of protein function in catalysis,7 T cell
activation,8 redox regulation,9-11 and signaling.10,12 Cysteine
sulfenic acid is also a precursor to both inter- and intramolecular
disulfides, as well as higher oxidation states of cysteine that can
influence the folding and consequently the function of the
protein.3 Additionally, cysteine sulfenic acid serves as a
biomarker for oxidative stress and occurs with high incidence in
certain types of cancer.13 Because of these diverse
——— ∗Corresponding author. Tel.: +61-8-8201-2268; e-mail:
[email protected]
ARTICLE INFO ABSTRACT
Article history: Received Received in revised form Accepted
Available online
Cysteine residues on proteins can react with cellular oxidants
such as hydrogen peroxide. While this process is important for
scavenging excess reactive oxygen species, the products of this
oxidation may also mediate cell signalling. To understand the role
of cysteine oxidation in biology, selective probes are required to
detect and quantify its occurence. Cysteine oxidation products such
as sulfenic acids are sometimes unstable and therefore short-lived.
If such cysteine derivatives are to be analysed, rapid reaction
with the probe is required. Here we introduce norbornene
derivatives as probes for cysteine oxidation, and demonstrate their
ability to trap sulfenic acids. The synthesis of norbornene
derivatives containing alkyne or biotin affinity tagsare also
reported to facilitate the use of these probes in chemical biology
and proteomics.
Keywords: Cysteine Cysteine sulfenic acid Norbornene Oxidative
stress Chemical biology
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biological implications, it is becoming increasingly important
to identify what proteins contain cysteine residues susceptible to
oxidation and if they exist as functional cysteine sulfenic acids.
In doing so, information about cysteine oxidation may be revealed
that can help clarify its role in both healthy and diseased
cells.
Figure 2: A selection of molecules that contain functional
groups that can react with cysteine sulfenic acid. These core
structures can be modified to contain a fluorophore or an affinity
tag such as biotin to facilitate detection, analysis, and imaging
after they have reacted with proteins.
Several functional groups are known to react with cysteine
sulfenic acids on peptides and proteins (Fig. 2), but there is
still a need for probes that trap short-lived cysteine sulfenic
acid residues.3,14 Unlike some cysteine sulfenic acid residues that
are persistent and stabilised by the protein microenvironment,15
many are short-lived precursors to higher oxidation states or other
modifications. A comprehensive mapping of their biological function
is far from complete.3 Dimedone (2)1,16 and its derivatives17,18
are widely used probes to trap cysteine sulfenic acids by reaction
of the nucleophilic α-carbon with the sulfur atom of the sulfenic
acid. While dimedone benefits from high chemoselectivity, it reacts
relatively slowly with sulfenic acids. This limitation has prompted
the Carroll laboratory to study other 1,3-dicarbonyls and related
nucleophiles that react more rapidly with cysteine sulfenic
acid.19,20 Indeed, subtle structural modulation of the dimedone
core has led to remarkably effective probes with rate enhancements
over 100-fold relative to dimedone.19,20 Mechanistically distinct
probes such as 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl 3,
an electrophilic probe for cysteine sulfenic acid) is also slow to
react and suffers from cross-reactivity with other cellular
nucleophiles.21 Boronic acids and benzoxaboroles such as 4 are also
electrophiles that react with cysteine sulfenic acids, but this
process is reversible.22 While 4 may therefore be useful in the
reversible inhibition of functional cysteine sulfenic acids, it is
not suitable for proteomics applications that require a stable
linkage to the cysteine residue. The strained trans-cyclooctene (5)
and cyclooctyne derivative 6 (BCN) are two additional probes
recently introduced that trap sulfenic acids through a
cycloaddition, providing a stable sulfoxide adduct.23,24 The
cycloaddition is driven by the release of ring strain that promotes
rapid ligation to sulfenic acids—an important feature for trapping
short-lived cysteine sulfenic acids. Unfortunately, this strain may
also lead to off-target reactions which compromise the selectivity
of the probe.25,26 For instance, the thiol-yne reaction of cellular
thiols with strained cyclooctynes such as 6 and 7 may limit the
generality of this class of molecules in detecting sulfenic acids
specifically.25 Additionally, compounds such as 5-7 are challenging
to synthesise and, because of their strain, have limited shelf-life
(especially in solution). For these reasons, we considered
norbornene derivatives as alternative probes that would react
rapidly with cysteine sulfenic acid due to release of strain (Fig.
3), but not be so reactive that the shelf-life and off-target
reactions are concerns. Additionally, norbornene derivatives are
straightforward to prepare in a modular fashion (by the Diels-Alder
reaction, for instance) so the prospect of accessing functionalised
probes in short-order was also attractive. Finally, norbornene
compatibility with proteins has been established through its use in
several selective bioconjugation methods.27
Figure 3: The strained alkene of easily prepared norbornene
derivatives is proposed here as a trap for short-lived cysteine
sulfenic acids
The use of alkenes to trap cysteine sulfenic acids dates back to
a report by Benitez and Allison in which water soluble
cyclohexene
derivatives were used to inhibit an acyl phosphatase containing
a catalytically active cysteine sulfenic acid.16 This was the same
study in which dimedone was initially reported to react with
cysteine sulfenic acid.16 The use of norbornene specifically, as a
probe for cysteine
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sulfenic acid, was inspired by classic studies by Barton and
co-workers in trapping the sulfenic acid formed during the
thermally induced syn-elimination of the sulfoxides of
penicillin.28-30 In this study we extend this concept to the amino
acid cysteine and demonstrate that the short-lived sulfenic acid
formed from the oxidation of N-acetylcysteine with hydrogen
peroxide can be intercepted by cycloaddition with various
norbornene derivatives.
Results and Discussion
Norbornene derivative 8 was selected as the first candidate
probe for cysteine sulfenic acid. 8 contains two carboxylic acids
that render it fully water-soluble after treatment with 1
equivalent of sodium carbonate, allowing these experiments to be
carried out in aqueous media (H2O and D2O, Fig. 4A) without the
need for an organic co-solvent—an important consideration for
applications on biological samples. The model sulfenic acid was
generated in situ by the oxidation of N-acetylcysteine (9) with
hydrogen peroxide. Because the conversion of 9 to its disulfide 10
was very rapid (See page S3 in the Supplementary Information), the
hydrogen peroxide was added to 8 first and then a solution of
N-acetylcysteine (9) was added slowly and in a 3-fold excess in a
second step. This protocol (excess N-acetylcysteine added to the
solution of 8 and hydrogen peroxide) ensured that the norbornene
probe would have a chance to react with the intermediate sulfenic
acid before all of it was converted to the disulfide. The reaction
was incubated for up to 30 minutes at room temperature and then
analysed directly by 1H NMR spectroscopy and LC-MS. The pH was
measured to be 4.3 over the course of the reaction. Gratifyingly,
while the major product detected by 1H NMR and LC-MS analysis was
disulfide 10, the alkene in 8 was completely consumed in its
conversion to 11 (Fig. 4A and S4-S5). Because the cycloaddition of
the cysteine sulfenic acid with 8 can proceed on either face of the
alkene and the sulfur in sulfoxide 11 is a stereogenic centre,
there are four possible diasteromers that can be formed. All four
diastereomers could be at least partially resolved in the LC-MS
analysis and the observed mass spectra were consistent with the
calculated value for 11 (m/z = 360, ESI-). In control reactions, it
was confirmed by 1H-NMR spectroscopy that 8 reacted with neither
hydrogen peroxide nor N-acetylcysteine 9 alone and that all three
components were required to form 11 (S4-S5). In control experiments
analysed by LC-MS, trace amounts of thiol-ene product were observed
from the direct reaction of norbornene derivative 8 and
N-acetylcysteine 9 (S6-S7), but this product was apparently below
the limits of detection in the 1H-NMR analysis (S4-S5). This result
suggests that even though norbornene has less strain than
cyclooctyne derivatives, 8 is not entirely inert to direct reaction
with thiols (or thiyl radicals generated in the presence of
oxygen). With that said, the key sulfenic acid trapping in Fig. 4A
was much faster, as no thiol-ene product was detected by LC-MS
(S8). When a similar series of experiments was carried out with
norbornene derivative 12, the same outcomes were observed (Fig. 4B
and S9-S13), with complete consumption of the alkene probe observed
only when 12 and 9 were reacted in the presence of hydrogen
peroxide. The pH for the reaction in Fig. 4B was slightly higher
(4.6 over the course of the reaction). The key sulfoxide products
13 and 14 can each be formed as a mixture of 4 diastereomers, which
could be partially resolved during the LC-MS analysis (S12). The
only products observed by 1H-NMR and LC-MS analysis of the reaction
mixture from Fig. 4B were the disulfide 10 and the anticipated
products formed from trapping the sulfenic acid (sulfoxides 13 and
14, m/z = 316, ESI-). No thiol-ene reaction was observed under
these conditions.
Figure 4: Norbornene probes 8 and 12 can trap the sulfenic acid
intermediate formed upon oxidation of N-acetylcysteine with
hydrogen peroxide.
For comparison to other previously reported probes for cysteine
sulfenic acid, dimedone (2) and cyclooctyne 6 were each used in an
attempt to trap the cysteine sulfenic acid formed from the reaction
of 9 with hydrogen peroxide (Fig. 5). In the attempt with dimedone,
the only cysteine-derived product observed by 1H-NMR and LC-MS was
disulfide 10 (Fig. 5A and S14-S19). No evidence of 15 (the expected
product of the reaction of dimedone and the sulfenic acid derived
from 9) was discovered. This results suggests the intermediate
sulfenic acid formed in the reaction reacts so rapidly with 9 that
dimedone is unable to trap it. This result highlights one of the
limitations of dimedone, but it also illustrates how efficiently
the norbornene probes 8 and 12 react with the sulfenic acid to form
sulfoxide adducts 11, 13 and 14 (Fig. 4). In control experiments in
which dimedone was treated with hydrogen peroxide (and no cysteine
derivative), unreacted dimedone was the major product detected by
both 1H-NMR and LC-MS, though trace amounts of another product were
observed (S15-S17). While the product was not isolated, it has a
mass consistent with an oxidative dimerisation (S17) and was
detected by LC-MS in all experiments in which dimedone and hydrogen
peroxide were present in the same reaction mixture. In the
comparison to cycloctyne 6 (BCN, Fig. 5B), the result was also a
surprise: the product expected upon trapping the sulfenic acid (16)
was not observed. The only product detected by LC-MS was disulfide
10. This result may be the result of limited solubility of 6 in
aqueous media and the need to use a mixed solvent system of water
and DMSO. Precipitation of 6 throughout the course of the reaction
may also complicate analysis. An additional complication is the
direct reaction of 6 with 9 via the thiol-yne reaction.25 In a
control reaction in which 6 was treated with a 3-fold excess of 9
(and no peroxide), the thiol-yne reaction was indicated
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by LC-MS (S22), however this side reaction was not observed
under oxidative conditions (S23). Cyclooctyne 7 was also subjected
to similar experiments, but it also suffered from limited
solubility and appeared to decompose in the presence of hydrogen
peroxide (S24-S27), so it was not pursued further as a probe. The
thiol-yne reaction was also observed in the reaction of
N-acetylcysteine (9) with the cyclooctyne 7 (S27-S28). These
results corroborate concerns recently reported by others about the
off-target reactions with thiols when using strained alkynes in
chemical biology,19,25 especially when the intention is to use the
alkyne to identify a specific oxidation state of cysteine such as
the sulfenic acid.19
Figure 5: Using similar conditions to the experiments in Figure
4, neither dimedone (2) nor cyclooctyne 6 trapped the sulfenic acid
intermediate formed upon oxidation of N-acetylcysteine with
hydrogen peroxide: neither 15 nor 16 were detected by 1H-NMR or
LC-MS analysis.
While the results in Figure 4 were a promising lead, the pH was
not controlled in these experiments. Therefore these reactions were
modified and carried out in a sodium acetate buffer (200 mM) at pH
5.0. The experiment with dimedone was also repeated in the same
buffer (Fig. 6). The outcome was largely the same: norbornene probe
8 was completely consumed in the reaction with the sulfenic acid
formed from 9 and converted into the four diastereomers of
sulfoxide 11 (Fig. 6 and S29-S35). Some unreacted cysteine was
detected (S29-S30) and thiol-ene adduct was also observed by LC-MS
(S31-S35), but the major products in the key reaction in Fig. 6A
were 10 and 11. Importantly, control experiments also demonstrated
that norborene probe 8 reacted with neither p-toluenesulfinate nor
p-toluenesulfonate (a model sulfinate and sulfonate, respectively)
at pD 5.0, indicating selectivity for the sulfenic acid oxidation
state (S36-S37). In contrast, the experiment with dimedone did not
result in reaction with the sulfenic acid. Unreacted 9, disulfide
10 and unreacted dimedone were the major products detected by
1H-NMR and LC-MS analysis (S38-S45). Again, some oxidation of
dimedone was observed by reaction with hydrogen peroxide (S44). In
a separate control experiment, this oxidation of dimedone was shown
to continue slowly over 24 hours (S40). These buffered experiments
at pH 5.0 were not repeated with alkynes 6 or 7 due to
complications arising from their low solubility in the reaction
medium.
Figure 6: A. Under buffered conditions (pH 5.0, 200mM NaOAc),
norbornene 8 can trap the sulfenic acid intermediate formed upon
oxidation of N-acetylcysteine with hydrogen peroxide. B. Under the
same conditions, dimedone (2) did not trap the sulfenic acid
intermediate formed upon oxidation of N-acetylcysteine with
hydrogen peroxide.
While the results in Figures 4 and 6 encourage further
investigation of norbornene-based probes for cysteine sulfenic
acid, this particular model system was sensitive to pH (and pD).
When 8 was used to trap the sulfenic acid derived from
N-acetylcysteine in
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sodium phosphate buffer at pD 7.4, the norbornene probe did not
react (Fig. 7A and S46-S47). At pD 7.4, the oxidation of
N-acetylcysteine (9) is faster than at pD 5.0: full conversion to
the disulfide was observed at the higher pD (Fig 7A and S46-S47)
and only 60% conversion to the disulfide was observed at pD 5.0
(Fig 7B and S48-S49) over a period of 20 minutes. It is likely that
both steps in the disulfide formation (nucleophilic attack of 9 on
hydrogen peroxide and then nucleophilic attack of 9 on the
resulting sulfenic acid) are slower at pD 5.0 because there will be
a lower concentration of the more nucleophilic thiolate derived
from 9 at the lower pD. The sulfenic acid in this system would be
expected to be longer-lived at pD 5.0 and therefore be able to
react with the norbornene probes. In contrast, the sulfenic acid
formed at pD 7.5 is consumed so rapidly by the reaction with the
thiolate of 9 that the norbornene cannot intercept any of the
sulfenic acid intermediate. Importantly, dimedone was also unable
to trap the sulfenic acid derived from 9 at pD 7.4 (S50-S51), which
is consistent with a very short-lived sulfenic acid. An additional
factor that may be important is the pKa of the cysteine sulfenic
acid itself. For small molecule sulfenic acids, the pKa can vary
from 4-12, and on proteins for which it has been measured, the pKa
of cysteine sulfenic acid was 6-7.
3,14 The cycloaddition proposed in Figure 3 may require the
sulfenic acid rather than its conjugate base, the sulfenate
anion—another potential reason why the reaction in Figure 7A at pD
7.4 did not provide the desired product 11. In any case, it was
fortuitous that the reaction mixtures in Figure 4 were naturally
between pH 4-5 without added buffer—conditions that enabled the
detection of the key ligation that validated norbornene derivatives
as probes for transient cysteine sulfenic acids. We also note that
norbornene probe 8 was still inert to model sulfinates and
sulfonates (p-toluenesulfinate and p-toluenesulfonate) at pD 7.4
(S52-S53).
Figure 7: A. In pD 7.4 sodium phosphate buffer (200 mM),
norbornene probe 8 did not trap the sulfenic acid intermediate
formed upon oxidation of N-acetylcysteine with hydrogen peroxide.
Full conversion of N-acetylcysteine (9) to disulfide 10 was
observed by 1H-NMR at this pD and no reaction of 8 was evident. B.
The oxidation of N-acetylcysteine (9) is slower at pD 5.0 than pD
7.4: only 60% conversion to disulfide 10 was observed by 1H-NMR,
with the remaining material present as unreacted 9.
For the detection of cysteine sulfenic acid on proteins, the
norbornene probe would require a reporter group—functionality that
can be further labeled, visualised, and quantified during SDS-PAGE,
Western blots, and cell imaging. Terminal alkynes are one such
group because they can be selectively labeled with an
azide-containing fluorophore or affinity tag12 via the
copper-catalysed azide-alkyne cycloaddition.31,32 We therefore
prepared 19 by direct reaction of amine 18 with anhydride 17,
providing norbornene probe 19 in 91% yield in a single step from
commercially available starting materials. The simplicity with
which the reporter group could be ligated to the core norbornene
structure is a valuable feature, as other probes for cysteine
sulfenic acid often require multiple steps and tedious
purification.3,14,20 The resulting carboxylic acid in 19 was also
anticipated to enhance water solubility. Unfortunately, probe 19
was surprisingly unstable in pH 5 buffer and hydrolysed to 8 and 18
rather quickly, with 50% hydrolysis observed by 1H-NMR over 20
minutes (Fig. 8 and S54-S57). For a sample incubated in buffer for
24 hours, complete hydrolysis was observed (S56). This hydrolysis
is a liability for analytical techniques such as SDS-PAGE, Western
blots, and cell imaging that necessitate the reporter group remain
ligated to the protein throughout the analysis. We suspect that 19
is prone to hydrolysis because the adjacent carboxylic acid can
participate in the reaction as a nucleophilic or acid catalyst.
Analogous cases of this neighbouring group participation have been
reported for arylamides on the same norbornene core,33 so
apparently this phenomenon extends to the alkylamide in 19.
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Figure 8: A. A norbornene probe for cysteine sulfenic acid
containing an alkyne reporter group (19) was prepared in a single
step by the reaction of amine 18 with anhydride 17. B. Probe 19
hydrolysed spontaneously in water.
To avoid the hydrolysis problem associated with 19, an
alternative norbornene probe was prepared that did not contain the
neighboring carboxylic acid. Norbornene derivative 12 was therefore
coupled to N-hydroxysuccinimide (20) to form NHS ester 21. Reaction
of 21 with amine 18 provided the new probe 22 (Fig. 9A). The amide
in this probe was stable and no hydrolysis was observed in
DMSO-water mixtures. Using a similar synthetic scheme, a
biotin-tagged norbornene was also synthesised (Fig. 9B). NHS ester
21 was first coupled to the diamine linker 23 in 91% yield.
Separately, biotin (25) was converted to its NHS ester 26. The free
amine in 24 was then used as a point of ligation for the biotin NHS
ester 26. After the final coupling, the target probe 27 was
isolated in 71% yield. Regarding solubility of the probes, a
solution of either 22 or 27 in DMSO can be added to phosphate
buffer such that the final concentration is 1 mM in 22 or 27, with
less than 1% of the total volume as DMSO. No precipitation was
observed and such formulations are comparable to other probes used
in pull-down assays. Norbornene derivatives 22 and 27 are now under
investigation as probes for cysteine sulfenic acids on both
proteins and cells.
Figure 9: A. A norbornene probe for cysteine sulfenic acid
containing an alkyne reporter group (22) was prepared in two steps
from 12. Probe 22 was not susceptible to hydrolysis in the same way
as 19. B. The synthesis of a norbornene probe for cysteine sulfenic
acid containing a biotin affinity tag.
Conclusions
A current challenge in the study of cysteine redox chemistry is
to detect rapid oxidation events and short-lived cysteine sulfenic
acids. The oxidation of N-acetylcysteine to its corresponding
disulfide is fast and therefore a challenging model system for any
probe designed to intercept the sulfenic acid intermediate.
Norbornene derivatives such as 8 and 12 were able to trap the
cysteine sulfenic acid
O
O
O
H
H
H2N
MeCN, 20 min, rtCO2H
O NH
91%
17
18
19
A.
B.
CO2H
O NH
19
pH 5NaOAc buffer
CO2H
O NH
19
CO2HCO2H
H2Nrt
8 18
+ +
~50% hydrolysis (NMR) after 20 min100% hydrolysis (NMR) after 24
hr
CO2H
12
H2NN OO
OH
20DCC, THF
rt
O
ON
O
O69%21
A.
18O
NH22i-Pr2NEt, CH2Cl2
89%
B.O
ON
O
O21
rt, 1h
H2NO
ONH2
i-Pr2NEt, CH2Cl2rt, 30min
23O
NH 24
OO
NH2
HN
NH
S
O
O
OH
H
N
O
OHN
NH
S
O
OH
OH
H 57%
N OO
OH
20
DCC, DMFrt25
91%
26
HN
NH
S
O
NH
OH
H
OO
NH
OH
71%27
24 + 26
i-Pr2NEt, DMFrt, 1h
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intermediate, providing stable sulfoxide adducts that could be
analysed by LC-MS. The most widely used probe for cysteine sulfenic
acid, dimedone, was unable to react with the transient sulfenic
acid formed in this model system. The direct reaction between
N-acetylcysteine (9) and the norbornene derivatives was observed,
but this off-target thiol-ene reaction was much slower than the
reaction between the norbornene derivatives and the cysteine
sulfenic acid. Furthermore, the product of the thiol-ene and the
sulfoxide formed from trapping the cysteine sulfenic acid have
distinct masses—an important point that will facilitate proteomics
applications of these probes. In the synthesis of a norbornene
derivative containing an alkyne reporter group (19), a surprisingly
fast amide hydrolysis reaction was discovered that cleaved the
reporter from the norbornene core structure. Revising the synthetic
strategy, alkyne and biotin labeled probes 22 and 27 were prepared
in short order; these compounds did not suffer the hydrolysis that
occurred for 19. With the functionalised norbornene probes in hand,
we are currently evaluating them in a variety of studies on
proteins and live cells with an aim to identify hitherto unknown
sites and functions of cysteine sulfenic acid.
Experimental section
For additional experimental details, control experiments, LC-MS
data and NMR spectra, please consult the Supplementary Information.
General considerations All reagents were used directly from
commercial suppliers without further purification. All reactions
without water as a solvent were carried out under an inert
atmosphere of nitrogen in flame-dried glassware. CH2Cl2 was
distilled over CaH2 and THF was distilled over sodium and
benzophenone prior to use. All other solvents were used directly
from commercial suppliers without further purification. Analytical
thin layer chromatography was performed on aluminium sheets coated
with silica gel containing a fluorescent indicator (0.15-0.2mm
thickness, 8 µm granularity), with visualisation carried out using
an ultraviolet lamp (254 nm) and/or development with potassium
permanganate. Column chromatography was performed using silica gel
(230–400 mesh, 60Å pore diameter). High resolution mass spectra
(HRMS) were recorded on a Waters Synapt HDMS Q-ToF by electrospray
ionization (ESI) or a Perkin Elmer, AxION, DSA-ToF by
atmospheric-pressure chemical ionization (APCI) and are reported as
the observed molecular ion. Infrared spectra were recorded on an
FTIR spectrometer with the absorptions reported in wavenumbers
(cm−1).
1H and 13C NMR spectra were recorded on a Bruker 600 MHz
Spectrometer. NMR were assigned using COSY, HMQC and HMBC where
required. Where D2O was used as the solvent and internal lock,
spectra were referenced to residual solvent for HOD (δH 4.79 ppm)
for 1H NMR. For MeOD-d4, spectra were referenced to residual
solvent (δH 3.33 ppm) for
1H NMR and (δC 49.0 ppm) for 13C
NMR. For DMSO-d6, spectra were referenced to residual solvent
(δH 2.50 ppm) for 1H NMR and (δC 39.5 ppm) for
13C NMR. For CDCl3, spectra were referenced to residual CHCl3
(δH 7.26 ppm) for
1H NMR and (δC 77.0 ppm) for 13C NMR. Chemical shift values
are reported in parts per million, 1H-1H coupling constants are
reported in hertz and H multiplicity is abbreviated as; s =
singlet, d = doublet, t = triplet, q = quartet, p = pentet, m =
multiplet, br = broad signal. Liquid chromatography mass
spectrometry (LC-MS) analyses were carried out on a Waters Acquity
UPLC coupled to a Micromass Quattro Micro triple quadrupole mass
spectrometer using electrospray ionization in both positive and
negative mode, as specified. A kinetex C18 column with particle
size 2.6 µm, and dimensions 50 × 2.1 mm length was used for all
experiments. UHPLC grade solvents and milli Q water were used in
these experiments. Before injection, samples were diluted to
approximately 50 µg/mL in milli Q water, mixed, and then filtered
through 0.2 µm nylon syringe filters. For all experiments, the
mobile phases were run with water 0.1 % formic acid (solvent A) and
acetonitrile (solvent B). All samples had an injection volume of 2
µL and ran with a flow rate of 0.2 ml min-1. The gradient was
programmed as followed: 95% A – 5% B (0 minutes) and maintained for
5 minutes (isocratic). 5% A – 95% B over 7 minutes and maintained
at 5% A for a further 3 minutes. 95% A – 5% B over 0.5 minutes and
held for a further 4.5 minutes to wash/equilibrate the column. The
total run time was 20 minutes. The electrospray source was operated
with a capiliary voltage of 3 kV and cone voltage of 30 V. The
source temperature was operated at 80 °C and the desolvation
temperature of 350 °C. All data was analysed using Masslynx
software. cis-5-norbornene-endo-2,3-dicarboxylic acid (8) as a
probe for cysteine oxidation (Figure 4A). Two solutions were first
prepared. Solution 1: In a vial, a mixture of
cis-5-norbornene-endo-2,3-dicarboxylic acid 8 (45 mg, 0.25 mmol),
D2O (2 mL) and Na2CO3 (25 mg, 0.24 mmol) was stirred or shaken
until fully dissolved. Solution 2: In a vial, a mixture of
N-acetylcysteine (125 mg, 0.77 mmol), D2O (2 mL) and Na2CO3 (25 mg,
0.24 mmol) was stirred or shaken until fully dissolved. For the key
reaction, solution 1 (0.4 mL, 0.05 mmol 8) was added to a vial,
followed by H2O2 (20 µL, 30 wt% in H2O) and stirred for a few
seconds before adding solution 2 (0.4 mL, 0.15 mmol, 9) dropwise
over 1 minute. The mixture was stirred for 20 minutes before
analysing directly by NMR. Full consumption of the alkene signal (δ
= 6.24 ppm) was observed. This reaction was repeated using H2O in
place of D2O and analysed by LC-MS. Disulfide 10 was detected at
2.53 min and the four diastereomers of 11 were detected at 3.05,
3.31, 3.49 and 4.19 minutes. exo-5-norbornenecarboxylic acid (12)
as a probe for cysteine oxidation (Figure 4B).
exo-5-Norbornenecarboxylic acid (12, 30 mg, 0.22 mmol) was added to
a vial and suspended in D2O (0.4 mL). Sodium carbonate (23 mg, 0.22
mmol) was then added and the mixture was stirred to provide a
homogeneous solution. Next, hydrogen peroxide (50 µL of a 30 wt%
solution in H2O, 0.44 mmol) was added to the solution of 12. In a
separate vial, N-acetylcysteine (9, 108 mg, 0.66 mmol) and sodium
carbonate (23 mg, 0.22 mmol) were dissolved in D2O (0.4 mL). The
solution of 9 was then added dropwise by pipette over 1 minute at
room temperature to the solution of 12. The reaction was stirred
for 30 minutes and then analysed by NMR. Full consumption of the
alkene signal (δ = 6.10 ppm) was observed. This reaction was
repeated using H2O in place of D2O and analysed by LC-MS. Disulfide
10 was detected at 2.52 min and the diastereomeric mixture of 13
and 14 were detected at 1.8-2.2, 3.4, 3.5, 3.7 and 3.8 minutes.
-
MAN
USCR
IPT
ACCE
PTED
ACCEPTED MANUSCRIPTTetrahedron 8
Dimedone as a probe for cysteine oxidation (Figure 5A). Two
solutions were first prepared. Solution 1: In a vial, a mixture of
dimedone (35 mg, 0.25 mmol), D2O (2 mL) and Na2CO3 (36 mg, 0.34
mmol) was stirred or shaken until fully dissolved. Solution 2: In a
vial, a mixture of N-acetylcysteine (211 mg, 1.30 mmol), D2O (3.2
mL), and Na2CO3 (54 mg, 0.50 mmol) was stirred or shaken until
fully dissolved. For the key reaction, solution 1 (0.4 mL, 0.05
mmol dimedone) was added to a vial, followed by H2O2 (20 µL, 30 wt%
in H2O). After a few seconds of stirring, solution 2 (0.4 mL, 0.16
mmol 9) was added dropwise over 1 minute. The solution was stirred
for 20 minutes before analysing directly by NMR. Only unreacted
dimedone and disulfide 10 were observed by 1H-NMR. This reaction
was repeated using H2O in place of D2O and analysed by LC-MS.
Disulfide 10 was detected at 2.61 min and unreacted dimedone at
7.34 minutes. 15 was not detected. cyclooctyne 6 as a probe for
cysteine oxidation (Figure 5B). Two solutions were first prepared.
Solution 1: In a vial, 6 (20 mg, 0.13 mmol) was dissolved in DMSO
(0.4 mL) and then D2O (1.6 mL) and Na2CO3 (15 mg, 0.14 mmol) were
added and the mixture was stirred or shaken until fully dissolved.
Note: BCN has very low solubility in aqueous media. Solution 2:
N-acetylcysteine (60 mg, 0.37 mmol), D2O (2 mL) and Na2CO3 (15 mg,
0.14 mmol) were added to a vial and stirred or shaken until fully
dissolved. For the key reaction, solution 1 (0.4 mL, 0.026 mmol 6)
was added to a vial, followed by H2O2 (20 µL, 30 wt% in H2O). After
stirring for a few seconds, solution 2 (0.4 mL, 0.074 mmol 9) was
added dropwise over 1 minute. The solution was stirred for 20
minutes before analysing directly by NMR. Only unreacted 6 and
disulfide 10 were observed. This reaction was repeated using H2O in
place of D2O and analysed by LC-MS. Only disulfide 10 was detected.
No sulfoxide adduct 16 was detected.
cis-5-norbornene-endo-2,3-dicarboxylic acid (8) as a probe for
cysteine oxidation at pD or pH 5.0 (Figure 6A). Two solutions were
first prepared. Solution 1: cis-5-norbornene-endo-2,3-dicarboxylic
acid (17 mg, 0.09 mmol 8), D2O (1.0 mL), and NaOH (3.8 mg, 0.09
mmol) were added to a vial and stirred or shaken until dissolved.
Finally, pD 5.0 acetate buffer in D2O (400 mM, 1.0 mL) was added
and the solution was stirred. Solution 2: N-acetylcysteine (42 mg,
0.26 mmol 9), D2O (1.0 mL), and NaOH (3.3 mg, 0.08 mmol) were added
to a vial and shaken or stirred until fully dissolved. Finally, pD
5.0 acetate buffer in D2O (400 mM, 1.0 mL) was added and the
solution was stirred. For the key reaction, solution 1 (0.5 mL,
0.02 mmol 8) was added to a vial followed by H2O2 (7 µL, 30 wt% in
H2O). The resulting solution was stirred for a few seconds before
the dropwise addition of solution 2 (0.5 mL, 0.06 mmol 9) over 1
minute. The solution was stirred for 20 minutes before analysing
directly by NMR. Full consumption of the alkene signal (δ = 6.25
ppm) was observed. This reaction was repeated using H2O in place of
D2O in a pH 5.0 NaOAc buffer and analysed by LC-MS. Disulfide 10
was detected at 2.50 minutes and four diastereomers of 11 were
detected at 2.97, 3.16, 3.39 and 4.04 minutes. The product of the
thiol-ene side reaction was detected at 7.36 minutes. Dimedone as a
probe for cysteine oxidation at pD or pH 5.0 (Figure 6B). Two
solutions were first prepared. Solution 1: Dimedone (13 mg, 0.09
mmol), D2O (1.0 mL) and NaOH (3.4 mg, 0.08 mmol) were added to a
vial and stirred or shaken until fully dissolved. Finally, pD 5.0
acetate buffer in D2O (400 mM, 1.0 mL) was added and the solution
was stirred. Solution 2: N-acetylcysteine (42 mg, 0.26 mmol), D2O
(1.0 mL) and NaOH (3.3 mg, 0.08 mmol) were added to a vial and
stirred or shaken until fully dissolved. Finally, pD 5.0 acetate
buffer in D2O (400 mM, 1.0 mL) was added and the solution was
stirred. For the key reaction, solution 1 (0.5 mL, 0.02 mmol) was
added to a vial followed by H2O2 (7 µL, 30 wt% in H2O) and stirred
for a few minutes before dropwise addition of solution 2 (0.5 mL,
0.06 mmol). The solution was stirred for 20 minutes before
analysing directly by NMR. Unreacted dimedone, unreacted
N-acetylcysteine and disulfide 10 were the only products observed.
15 was not detected. This reaction was repeated using H2O in place
of D2O in a pH 5.0 NaOAc buffer and analysed by LC-MS. Disulfide 10
was detected at 2.51 minutes and unreacted dimedone at 7.37
minutes. 15 was not detected.
cis-5-norbornene-endo-2,3-dicarboxylic acid (8) as a probe for
cysteine oxidation at pD or pH 7.4 (Figure 7A). Two solutions were
first prepared. Solution 1: cis-5-norbornene-endo-2,3-dicarboxylic
acid (20.0 mg, 0.11 mmol 8), D2O (1.25 mL) and NaOH (5 mg, 0.12
mmol) were added to a vial and stirred or shaken until fully
dissolved. Finally, additional pD 7.4 sodium phosphate buffer (400
mM in D2O, 1.25 mL) was added and the solution was stirred.
Solution 2: N-acetylcysteine (49.8 mg, 0.30 mmol 9), D2O (1.25 mL)
and NaOH (3 mg, 0.08 mmol) were added to a vial and stirred until
fully dissolved. Finally, additional pD 7.4 sodium phosphate buffer
(400 mM in D2O, 1.25 mL) was added and the solution was stirred.
For the key reaction, solution 1 (0.5 mL, 0.02 mmol 8) was added to
a vial followed by H2O2 (7 µL, 30 wt% in H2O). The solution was
stirred for a few minutes before adding solution 2 (0.5 mL, 0.06
mmol 9) dropwise over 1 minute. The solution was stirred for 20
minutes before analysing directly by NMR. The only products
observed were unreacted 8 and disulfide 10. 11 was not detected.
Oxidation of N-acetylcysteine to its disulfide using hydrogen
peroxide at pD 5.0 (Figure 7B). N-acetylcysteine (9.8 mg, 0.06 mmol
9), D2O (0.25 mL) and NaOH (2.4 mg, 0.06 mmol) were added to a vial
and stirred or shaken until fully dissolved. Next, pD 5.0 sodium
acetate buffer (400 mM in D2O, 0.25 mL) was added and the solution
was stirred. A solution of H2O2 (7 µL, 30 wt% in H2O) was added and
left to stir for 20 min. The reaction was then analysed directly by
1H-NMR. 60% conversion to disulfide 10 and 40% unreacted 9 were
observed. Synthesis of alkyne 19 (Figure 8A). 1-amino-3-butyne
(59.2 µL, 0.723 mmol) was added to a stirred solution of
cis-5-norbornene-endo-2,3-dicarboxylic anhydride (239 mg, 1.46
mmol) in acetonitrile (1 mL) and stirred at room temperature for 20
minutes, over which time a white precipitate formed. The resulting
mixture was transferred into a centrifuge tube and pelleted by
centrifugation for 10 minutes. The supernatant was decanted and the
remaining pellet washed with EtOAc. The final product was isolated
by filtration without further purification to give the product 19
as a white solid (154 mg, 91% yield): m.p. 129 ºC; IR (νmax, ATR):
3359, 2987, 1716, 1622, 1550, 1321, 1267, 1229, 1074, 846, 759,
679, 625; 1H NMR (600 MHz, DMSO-d6): δ = 11.53 (1H, br-s, COOH),
7.86 (1H, t, J =
-
MAN
USCR
IPT
ACCE
PTED
ACCEPTED MANUSCRIPT 9
5.9 Hz, NH), 6.15 (1H, dd, J = 5.5, 2.9 Hz, HC=CH), 5.95 (1H,
dd, J = 5.4, HC=CH), 3.14 (1H, dd, J = 10.3, 3.3 Hz, CH=CHCHCHCOOH
or CH=CHCHCHCONH), 3.08 (3H, contains CONHCH2CH2 and CH=CHCHCHCOOH
or CH=CHCHCHCONH), 2.93 (2H, m, CHCH=CHCH), 2.80 (1H, t, J = 2.6,
C≡CH), 2.20 (2H, m, CH2C≡CH), 1.25 (1H, d, J = 8.1 Hz, CHCHAHBCH),
1.21 (1H, d, J = 8.2 Hz, CHCHAHBCH);
13C NMR (150 MHz, DMSO-d6): 173.5, 171.2, 134.9, 133.7, 82.4,
71.9, 48.4, 48.1, 48.1, 46.7, 45.3, 37.8, 18.7; LRMS (ESI): [M-H]
-: found 232.0, C13H14NO2 requires 232.1 Hydrolysis study of alkyne
probe 19 (Figure 8B). Alkyne probe 19 (24 mg, 0.10 mmol) was added
to a vial and dissolved in 1.25 mL of D2O. To this solution was
added sodium acetate buffer in D2O (1.25 mL, pD 5.0, 400 mM). After
20 minutes and 24 hours, this solution was analysed directly by 1H
NMR and LC-MS, indicating hydrolysis to 8 and 18. After 20 minutes,
50% hydrolysis was observed by 1H-NMR and complete hydrolysis was
observed after 24 hours (as indicated by integration of the alkene
signals and comparison to an authentic sample of 8, 19, and 18).
Norbornene NHS derivative 21. Exo-5-norbornenecarboxylic acid (455
mg, 3.30 mmol), N-hydroxysuccinimide (357 mg, 3.40 mmol), and
N,N’-dicyclohexylcarbodiimide (679 mg, 3.30 mmol) were dissolved in
anhydrous THF (15 mL) and stirred at room temperature overnight,
over which time a white precipitate formed (N,N’-dicyclohexylurea).
The precipitate was removed by filtration and washed with THF and
the filtrate was collected and concentrated under reduced pressure.
The crude solid was purified by column chromatography (40% EtOAc in
hexane) to give the product 21 as a white solid (537 mg, 69 %).
m.p. 84-86°C; Rf (40% EtOAc:hexane) 0.37; IR (νmax, ATR): 2983,
2949, 1735, 1428, 1361, 1200, 1047, 947, 841, 711, 644 cm
–1; 1H NMR (600 MHz, CDCl3): δ = 6.20 (1H, m, CH2CHCH=CH), 6.15
(1H, m, CHCH=CH), 3.28 (1H, m, CH=CHCHCH), 3.00 (1H, m,
CH=CHCHCH2), 2.83 (4H, m, COCH2CH2CO), 2.51 (1H, m, COCHCHAHB),
2.05 (1H, m, COCHCHAHB), 1.58-1.50 (2H, contains COCHCHAHBCH and
COCH), 1.45 (1H, CHCHAHBCH);
13C NMR (150 MHz, CDCl3): δ = 171.6, 169.3, 138.5, 135.3, 47.1,
46.4, 41.8, 40.3, 31.0, 25.6. Norbornene probe 22. Norbornene-NHS
21 (359 mg, 1.53 mmol) was dissolved in anhydrous DCM (8 mL) with
stirring at room temperature and then 1-amino-4-butyne (125 µL,
1.53 mmol) and DIPEA (650 µL, 3.82 mmol) were then added
successively. After 1 hour a white precipitate had formed
(N-hydroxysuccinimide). The crude material was concentrated under
reduced pressure and purified by column chromatography (40 % EtOAc
in hexane) to give the product 22 as a white solid (257 mg, 89 %).
m.p. 86-88 °C; Rf (40 % EtOAc in hexane) 0.47; IR (νmax, ATR) 3300,
3267, 3058, 2963, 2869, 1633, 1547, 1442, 1359, 1330, 1243, 1221,
1149, 1070, 1018, 901, 864, 721, 680, 625 cm–1; 1H NMR (600 MHz,
CDCl3): δ = 6.15 (1H, dd, J = 5.7, 3.0 Hz, CH=CH), 6.11 (1H, dd, J
= 5.7, 3.0 Hz, CH=CH), 5.81 (1H, br-s, CONH), 3.42 (2H, m,
CONHCH2CH2), 2.93 (2H, m, CHCH=CHCH), 2.42 (2H, tdd, J = 6.3, 2.7,
1.6 Hz, CONHCH2CH2), 2.01 (2H, contains CH2C≡CH and CH=CHCHCHCONH),
1.92 (1H, m, CH=CHCHCHAHB), 1.70 (1H, dd, J = 8.4, 1.7 Hz,
CHCHAHBCH), 1.35 (2H, contains CHCHAHBCH and CH=CHCHCHAHB);
13C NMR (150 MHz, CDCl3): δ = 175.6, 138.3, 136.0, 81.7, 69.9,
47.2, 46.4, 44.8, 41.6, 38.0, 30.5, 19.5; HRMS (ESI): [M+H]+, found
190.1228. C12H16NO
+ requires 190.1226. Norbornene amine derivative 24.
1,2-bis(2-aminoethoxy)ethane (624 µL, 4.2 mmol) and DIPEA (163 µL,
0.92 mmol) were dissolved in anhydrous DCM (5 mL) with stirring at
room temperature. A solution of norbornene-NHS 21 (99 mg, 0.42
mmol) was dissolved in anhydrous DCM (1 mL) and added dropwise to
the 1,2-bis(2-aminoethoxy)ethane solution and then the reaction
mixture was stirred at room temperature for 30 min. After this
time, the crude mixture was concentrated under reduced pressure.
The resulting oil was purified by column chromatography (10% MeOH
in DCM with 1% NEt3) to give the product 24 as a yellow oil (103
mg, 91 %). Rf (10% MeOH in DCM with 1% NEt3) 0.20; IR (νmax, ATR):
3294, 3057, 2936, 2868, 1645, 1541, 1448, 1351, 1247, 1105, 905,
808, 724 cm
–
1; 1H NMR (600 MHz, CDCl3): δ = 6.29 (1H, br-s, CONH), 6.13 (1H,
dd, J = 5.7, 2.9 Hz, CH=CH), 6.09 (1H, dd, J = 5.8, 3.1 Hz, CH=CH),
3.63 (4H, m, 2 x CH2 PEG), 3.56 (4H, dt, J = 18.5, 5.1 Hz, 2 x CH2
PEG), 3.47 (2H, q, J = 5.2 Hz, CONHCH2), 2.91 (4H, m, contains CH2
PEG and CHCH=CHCH), 2.09 (1H, m, CHCHCONH), 1.91 (1H, dt, J = 11.5,
4.0 Hz, CH=CHCHCHAHB), 1.72 (1H, d, J = 8.3 Hz, CHCHAHBCH), 1.31
(2H, contains CH=CHCHCHAHB and CHCHAHBCH);
13C NMR (150 MHz, CDCl3): δ = 175.8, 138.2, 136.0, 72.9, 70.2,
70.1, 70.0, 47.2, 46.3, 44.6, 41.6, 39.3, 30.5; HRMS (ESI): [M+H]+,
found 269.1867. C14H25N2O3
+ requires 269.1865. Biotin NHS ester 26. Biotin (498 mg, 2
mmol) was dissolved in anhydrous DMF (10 mL) by heating to
approximately 70 °C for 10 minutes or until fully dissolved. The
reaction mixture was allowed to cool to room temperature before
adding N-hydroxysuccinimide (240 mg, 2.1 mmol) with stirring at
room temperature. A solution of N,N’-dicyclohexylcarbodiimide (438
mg, 2.13 mmol) in anhydrous DMF (2 mL) was added dropwise to the
stirred solution. The reaction was then stirred overnight at room
temperature over which time a white precipitate formed
(N,N’-dicyclohexylurea). The precipitate was removed by filtration
and washed with DMF. The filtrate was diluted with EtO2 until a
white precipitate formed. The precipitate was collected by
filtration and rinsed with EtO2 then dried to give the crude
product 26 as a white solid (395 mg, 57 %): m.p. (decomp.) 178–190
°C; IR (νmax, ATR): 3227, 2941, 2876, 1818, 1788, 1729, 1698, 1465,
1369, 1210, 1071, 861, 739, 656 cm–1; 1H NMR (600 MHz, DMSO-d6): δ
= 6.43 (1H, s, NH), 6.37 (1H, s, NH), 4.30 (1H, m, HNCHCHNH), 4.14
(1H, m, HNCHCHNH), 3.10 (1H, m, SCH), 2.84-2.78 (5H, m, contains
NCOCH2CH2 and SCHAHB), 2.67 (2H, t, J = 7.7 Hz, CH2CH2CO2N), 2.57
(1H, d, J = 12.4 Hz, SCHAHB), 1.64 (3H, contains
CHAHBCH2CH2CH2CO2N), 1.52-1.36 (3H, m, contains
CHAHBCH2CH2CH2COON);
13C NMR (150 MHz, DMSO-d6): δ = 170.3, 169.0, 162.7, 61.0, 59.2,
55.3, 40.1 (overlaps with NMR solvent peak), 30.0, 27.9, 27.6,
25.5, 24.3; HRMS (ESI): M+H+, found 342.1128. C14H20N3O5S
+ requires 342.1118. Norbornene probe 27. Biotin-NHS 27 (382 mg,
1.12 mmol) was dissolved in anhydrous DMF (6 mL) with stirring at
50 °C for 5 minutes or until dissolved. Norbornene derivative 24
(398 mg, 1.5 mmol) was dissolved in anhydrous DMF (2 mL) and added
dropwise to the biotin-NHS solution. DIPEA (240 µL, 1.4 mmol) was
added and the reaction stirred at room temperature for 1 hr. Upon
completion, EtO2 was added until a white precipitate formed. The
precipitate was collected by filtration and washed with additional
EtO2. The resulting residue was purified by column chromatography
(10% MeOH in DCM) to give the product 27 as a white solid (393 mg,
71%): m.p. 126-131 °C; Rf (10 % MeOH in DCM) 0.32; IR (νmax, ATR):
3291, 2942, 2869, 1703, 1639, 1551, 1464, 1309, 1248,
-
MAN
USCR
IPT
ACCE
PTED
ACCEPTED MANUSCRIPTTetrahedron 10
1214, 1129, 987, 867, 724 cm–1; 1H NMR (600 MHz, MeOD): δ = 6.16
(2H, m, CHCH=CHCH), 4.51 (1H, dd, J = 8.3, 4.5 Hz, CONHCHCH2S),
4.32 (1H, dd, J = 7.9, 4.4 Hz, CONHCHCHS), 3.64 (4H, m, 2 x CH2
PEG), 3.57 (4H, m, 2 x CH2 PEG), 3.39 (4H, m, 2 x CH2 PEG), 3.23
(1H, m, SCH), 2.95 (1H, dd, J = 12.8, 5.0 Hz, CONHCHCHAHBS), 2.89
(2H, m, CHCH=CHCH), 2.73 (1H, d, J = 12.7 Hz, CONHCHCHAHBS), 2.24
(2H, t, J = 7.1 Hz, CH2CONH), 2.15 (1H, m, CH=CHCHCH), 1.88 (1H,
dt, J = 11.7, 4.0 Hz, CH=CHCHCHAHB), 1.79-1.59 (5H, contains 2 x
CH2 Biotin and CHCHAHBCH), 1.46 (2H, p, J = 7.5 Hz, CH2 Biotin),
1.33 (2H, contains CH=CHCHCHAHB and CHCHAHBCH);
13C NMR (150 MHz, MeOD): δ = 178.6, 176.2, 166.1, 139.0, 137.3,
71.3, 70.7, 70.6, 63.4, 61.6, 57.0, 48.6, 47.1, 45.2, 42.8, 41.0,
40.4, 40.3, 36.7, 31.2, 29.8, 29.5, 26.8; HRMS (ESI): M+H+, found
495.2641. C24H39N4O5S
+ requires 495.2636.
Acknowledgments
The authors acknowledge generous financial support from the
Australian Research Council (DE150101863, J.M.C), The Royal Society
(University Research Fellowship, G.J.L.B), the EPSRC (G.J.L.B) and
the European Research Council (Starting Grant, G.J.L.B.). Daniel
Jardine of Flinders Analytical is acknowledged for assistance with
mass spectrometry.
References and notes
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Supplementary Material
Supplementary material is available, including additional
experimental details, NMR spectra, and LC-MS data.